Infants fed synthetic formulas suffer a higher incidence of iron-deficiency anemia and infection than do infants that are breast fed. As a result, there are plans to supplement formulas with natural constituents of milk that may be responsible for enhanced iron absorption and for anti-microbial activity. An extremely attractive candidate in this regard is lactoferrin(Lf), a molecule that has been implicated in both functions because of its extremely high avidity for iron. However, in spite of a compelling rationale, studies designed to test for the occurrence of these physiological activities of Lf in vivo have been inconsistent. Even when positive effects have been obtained, they have not necessarily pointed to a mechanism based on iron binding. The situation has been further complicated with the attribution to Lf of a wide array of additional, dissimilar functions, some of which appear to involve regulatory interactions with cells and also to be independent of iron-binding. It is the long-term objective of our laboratory to conclusively identify the in vivo physiological roles of Lf and to determine how they could be exploited for preventive or therapeutic purposes. our studies to date have revealed several novel aspects of Lf that could be germane to its in vivo activity: there exist multiple forms of Lf, at least one of which does not bind iron and, instead, exhibits a potent ribonuclease activity; Lf has antitumor activity which, importantly, occurs through activation of natural killer (NK) cells; and Lf can act directly to cause gene transcription in target cells. We believe that these findings offer a new perspective on Lf function an possibly an explanation for the disparate results obtained in prior studies. Our working hypothesis is that the broad functions proposed for Lf, and their inconsistent expression, are the result of the existence of these functionally distinct isoforms and the fact that they act, for some processes, through cells of the immune system. We propose to test this hypothesis directly in the context of Lf supplementation of infant formula for purposes of preventing infection. The studies will be carried out under 2 specific aims; 1. to determine the role of different isoforms in the anti-infective properties of Lf in milk; and 2. to determine the role of (gut associated and systemic) lymphoid cells in the anti-infective properties of Lf. We believe that the results of this work could provide an understanding of the effects and potential benefits of Lf supplementation of infant formula.